The synthesis, full characterization, photochemical properties, and cytotoxic activity toward cisplatin-resistant cancer cell lines of new semisquaraine-type Pt(II) complexes are presented. The synthesis of eight semisquaraine-type ligands has been carried out by means of an innovative, straightforward methodology. A thorough structural NMR and X-ray diffraction analysis of the new ligands and complexes has been done. Density functional theory calculations have allowed to assign the trans configuration of the platinum center. Through the structural modification of the ligands, it has been possible to synthesize some complexes, which have turned out to be photoactive at wavelengths that allow their activation in cell cultures and, importantly, two of them show remarkable solubility in biological media. Photodegradation processes have been studied in depth, including the structural identification of photoproducts, thus justifying the changes observed after irradiation. From biological assessment, complexes C7 and C8 have been demonstrated to behave as promising photoactivatable compounds in the assayed cancer cell lines. Upon photoactivation, both complexes are capable of inducing a higher cytotoxic effect on the tested cells compared with nonphotoactivated compounds. Among the observed results, it is remarkable to note that C7 showed a PI > 50 in HeLa cells, and C8 showed a PI > 40 in A2780 cells, being also effective over cisplatin-resistant A2780cis cells (PI = 7 and PI = 4, respectively). The mechanism of action of these complexes has been studied, revealing that these photoactivated platinum complexes would actually present a combined mode of action, a therapeutically potential advantage.
The synthesis, full characterization, photochemical properties, and cytotoxic activity toward cisplatin-resistant cancer cell lines of new semisquaraine-type Pt(II) complexes are presented. The synthesis of eight semisquaraine-type ligands has been carried out by means of an innovative, straightforward methodology. A thorough structural NMR and X-ray diffraction analysis of the new ligands and complexes has been done. Density functional theory calculations have allowed to assign the trans configuration of the platinum center. Through the structural modification of the ligands, it has been possible to synthesize some complexes, which have turned out to be photoactive at wavelengths that allow their activation in cell cultures and, importantly, two of them show remarkable solubility in biological media. Photodegradation processes have been studied in depth, including the structural identification of photoproducts, thus justifying the changes observed after irradiation. From biological assessment, complexes C7 and C8 have been demonstrated to behave as promising photoactivatable compounds in the assayed cancer cell lines. Upon photoactivation, both complexes are capable of inducing a higher cytotoxic effect on the tested cells compared with nonphotoactivated compounds. Among the observed results, it is remarkable to note that C7 showed a PI > 50 in HeLa cells, and C8 showed a PI > 40 in A2780 cells, being also effective over cisplatin-resistant A2780cis cells (PI = 7 and PI = 4, respectively). The mechanism of action of these complexes has been studied, revealing that these photoactivated platinum complexes would actually present a combined mode of action, a therapeutically potential advantage.
For decades, platinum
complexes have focused a great interest because
of their particular features.[1] In recent
years, these complexes have re-emerged as very motivating entities
for different purposes.[2] In particular,
photoresponsive complexes have become significant,[3] being one of the fields in cancer therapies where most
efforts have been invested.[4] In the last
years, platinum(II) phototherapy has been shown as an opportunity
to lessen the side effects associated to cisplatin-type drugs.[5] Roughly, phototherapy can be classified into
two categories: photodynamic therapy (PDT) and photoactivated chemotherapy
(PACT). Both PDT and PACT are proving to be good options as new cancer
therapies.[6] While PDT needs the presence
of molecular oxygen to elicit cell death, PACT acts by means of the
species generated by a prodrug as its photoresponse.[7] In this way, PACT allows controlling where and when the
active species are generated, selectivity is thus increased, and therefore,
the necessary dose is reduced. Such an approach allows minimizing
side effects, hence improving the quality of life of the treated patients.However, despite all the efforts being made, there are some aspects
that remain unsolved associated with the physical properties of the
platinum complexes themselves. One of these limitations is, for example,
the stability of the complexes in the physiological medium and the
dark. This refers not only to the photostability of the complex but
also to the resistance to hydrolysis.[8] The
solubility of platinum(II) species in aqueous media is another concern.
In general, neutral complexes have low solubility; however, in order
to make them work, they must have a certain solubility in physiological
media,[9] though keeping an equilibrium with
lipophilicity, as this feature is believed to be crucial for the passive
influx of drug molecules into cells.[10] Also,
the frequency of irradiation where a complex response occurs is critical,
as ideally, it should take place in the frequency range known as the
therapeutic window.[5e] In this article,
we want to present a new class of platinum(II) complexes and how we
overcome the limitations pointed out above. Thus, herein we report
the synthesis of new ligands and their corresponding platinum(II)
complexes and their complete photochemical characterization. Recently,
we have described the synthesis and photochemical assessment and activity
against HeLa cells of new squaramide-based platinum(II) complexes.[11] Although promising results were obtained, some
limitations were noticeable. Those stable complexes showed moderate
to low solubility in aqueous media and, importantly, all of them were
photoresponsive in the UV range, clearly out of the therapeutic window.
In the study herein reported, the aforementioned problems have been
alleviated. We believe that these new complexes may have applications
given their ability of photoresponse. The new ligands here presented
are semisquaraine-type (Figure ), and based on the conclusions of our previous study, they
bear a thioalkylamino branch as a cooperative cyclobutenedione substituent,
considering its chelation abilities. Also, unlike our former squaramide-type
ligands, the other substituent of the ring has been used to increase
electronic delocalization and, therefore, modify the absorption range
of these new complexes. Finally, several structural modifications
for the improvement of the solubility of the complexes are discussed.
Figure 1
New semisquaraine-type
Pt(II) complexes in this study.
New semisquaraine-type
Pt(II) complexes in this study.
Results
and Discussion
Synthesis
In view of our previous
findings, we planned
to synthesize a new series of cyclobutenedione-based platinum(II)
complexes fulfilling the following requirements: (i) bearing two 3-thiopropylamino
appendixes, since this kind of chelators proved to be efficient for
the Pt(II) release from the complex; (ii) enhanced water solubility,
to improve the availability in a physiological medium; and (iii) photochemical
response in a biocompatible frequency range. We visualized that the
attachment of a pyrrole linker to the cyclobutenedione core could
hopefully facilitate the accomplishment of these requirements since
such a subunit should be amenable to subtle structural modifications,
oriented to achieve the desired properties in the new photoactive
complexes. Thus, an accessory phenyl residue conjugated to the heterocycle
would extend the π delocalization, providing a chromophore suitable
for a low-frequency photoactivation, while the addition of hydroxyl
groups and their PEG derivatives would improve water solubility. Moreover,
pyrrole is considered a privileged scaffold for biomedical applications.[12] Accordingly, the synthesis of complexes C1–8 (Figure ) was undertaken.The first endeavor was the preparation
of the corresponding ligands, L1 to L8.
To this aim, we developed a one-pot protocol consisting of the consecutive
addition of the pyrrole and the methylthiopropylamino nucleophilic
moieties to the cyclobutenedione core. The proper conditions were
set up by investigating, in depth, the reaction depicted in Scheme . The pyrrole component 10 was prepared by N-alkylation of pyrrole according to a
known procedure.[13] Successive conjugate
addition of 10 (in a molar ratio 9/10 = 1/1) and then a mixture of thioamine 11 and
diisopropylethylamine (DIPEA) in a molar ratio 9/11/DIPEA = 1/1/2, to a solution of dichlorocyclobutenedione 9(14) in tetrahydrofuran (THF), furnished
the expected ligand L1 in a single operation and a 53%
isolated yield after crystallization from MeOH.
Scheme 1
Sequential Strategy
in the Synthesis of Ligand L1
Next, we prepared the required pyrrole derivatives for the synthesis
of the other targeted ligands (Scheme ). N-alkylation of 1H-pyrrole-2-carbaldehyde, 12, with (3-bromopropyl)(methyl)sulfane furnished aldehyde 13, which was subjected to a Horner–Wadsworth–Emmons
(H–W–E) alkenylation with the benzylic phosphonates 14(15) and 15,[16] affording the corresponding pyrrole derivatives 17 and 18, respectively, in good yields. It is
worth mentioning that alkene 17 showed a limited stability,
even at freezing temperatures, leading to degradation products after
several weeks. Hence, it should be soon subjected to the next synthetic
step. On the contrary, its congener 18 showed an unlimited
stability.
Scheme 2
Synthesis of Substituted Pyrrole Motives
When the conditions developed for the preparation
of L1 from the simpler pyrrole 10 were applied
to 17, after chromatographic purification followed by
crystallization
from CH2Cl2/Et2O, the expected ligand L2 was obtained as a yellow powder in a 60% yield (Scheme ). Moreover, it was
found that the same reaction performed in a two-phase CH2Cl2/H2O system allowed the isolation of pure L2 in a 72% yield by straight crystallization after the addition
of Et2O to the dried organic phase, without the need of
a previous chromatographic separation. This improved protocol applied
to pyrrole 18 furnished the corresponding ligand L4 as an orange powder in a 57% yield. Eventually, crystallization
of L2 from dimethylformamide (DMF)/H2O provided
crystal needles suitable for X-ray analysis (Figure ), which confirmed the 1,2-disubstitution
of the cyclobutenedione core, the E configuration
of the alkene, and a preferential fully extended conformation of the
two substituents, with the NH syn to the pyrrole
moiety.
Scheme 3
Synthesis of Conjugate Pyrrole Ligands L2–8
Figure 2
MERCURY drawing for L2. Thermal ellipsoids are drawn
at the 30% probability level. H atoms are omitted for clarity.
MERCURY drawing for L2. Thermal ellipsoids are drawn
at the 30% probability level. H atoms are omitted for clarity.Treatment of L2 and L4 with tribromoborane
in CH2Cl2 unveiled the hydroxyl groups, furnishing
the corresponding phenols L3 and L5 in good
yields. Phenol L3 was readily converted into the glycol
ether derivatives L6 and L8 in 47 and 92%
yield after crystallization from methanol and CH2Cl2/Et2O, respectively. However, when the same methodology
was intended for the synthesis of L7, the O-alkylation
of the pyrogallol derivative L5 failed. Alternatively, L7 was synthesized from pyrrole 19, prepared
through an H–W–E reaction between aldehyde 13 and phosphonate 16(17) (Scheme ). As 19 was revealed to be very unstable, for the synthesis of L7, freshly prepared crude 19 was added to cyclobutenedione 9, and after a successive addition of thioamine 11, the expected ligand was obtained in a 62% global yield from 13. Likewise, an alternative synthesis of L6 starting
from 13 and an appropriate phosphonate was also developed
(see the Supporting Information, S3 for
details), but the overall sequence yield did not improve the results
of the pathway shown in Scheme .All the synthesized ligands (L1–8) and their
previously unknown precursors (13, 17, and 18) were fully characterized according to their physical and
spectroscopic data.With the ligands in hands, the next challenge
was synthesizing
their Pt(II) complexes. To this aim, each ligand was treated with
K2PtCl4 in an appropriate solvent at room temperature
under an argon atmosphere. For complexes C1–6,
the resulting precipitate was filtered and successively washed with
suitable solvents. For C7 and C8, the complexes
were separated from the reaction mixture by the addition of brine,
followed by extraction with CH2Cl2. Table summarizes conditions,
yield, and physical description for each particular complex.
Table 1
Synthesis of Complexes C1–8a
entry
solvent
time (h)
precipitate washingb
complex (yield)
color, mp (from solvent)
1
MeOH/H2O, 1:1
17
MeOH; H2O
C1 (56%)
brown, 173–178 °C (MeOH/H2O)
2
THF/H2O, 3:2
22
THF; H2O
C2 (97%)
orange, 184–187 °C (THF/H2O)
3
THF/H2O, 1:1
15
H2O;
MeOH; THF
C3 (86%)
russet, >200 °C (THF/H2O)
4
THF/H2O, 1:1
20
THF; H2O
C4 (99%)
orange, >200 °C (THF/H2O)
5
THF/H2O, 1:1
16
H2O; MeOH; THF
C5 (85%)
brown, >200 °C (THF/H2O)
6
THF/H2O, 1:1
16
H2O; MeOH; THF
C6 (81%)
orange, 135–139 °C (THF/H2O)
7
THF/H2O, 1:1
17
c
C7 (65%)
brown, 92–95 °C (CH2Cl2/Et2O)
8
H2O
17
c
C8 (95%)
brown, 50–53 °C (CH2Cl2/Et2O)
All the reactions were performed
by the treatment of the starting ligands with 1 M equivalent of K2PtCl4 at room temperature, under an argon atmosphere,
except for entry 8, where the molar ratio K2PtCl4/L8 was 1.3.
Complexes precipitated from the
reaction mixture were filtered and successively washed with the indicated
solvents.
Complexes C7 and C8 were separated by extraction with CH2Cl2.
All the reactions were performed
by the treatment of the starting ligands with 1 M equivalent of K2PtCl4 at room temperature, under an argon atmosphere,
except for entry 8, where the molar ratio K2PtCl4/L8 was 1.3.Complexes precipitated from the
reaction mixture were filtered and successively washed with the indicated
solvents.Complexes C7 and C8 were separated by extraction with CH2Cl2.All the synthesized complexes C1–8 were fully
characterized according to their physical and spectroscopic data.
HRMS or MS of all the complexes showed the characteristic pattern
of platinum compounds except for C8, which degraded upon
ionization. Since its polydisperse polymeric nature precluded elemental
analysis, the amount of Pt was determined by ICP-OES (6.7% Pt), obtaining
a value similar to that expected, assuming a molecular weight of 2705
Da (7.2% Pt).The cis–trans configuration
of Pt(II) complexes usually reveals crucial for the biological response
to these species. Unfortunately, all attempts to obtain crystalline
complexes for their structural elucidation by means of X-ray diffraction
(XRD) analysis were unsuccessful. As tert-butyldiphenylsilyl
(TBDPS) derivatives are usually prone to form crystalline solids,
a TBDPS-derivative ligand L20 and its corresponding dichloride-Pt(II)
complex, C20, were prepared (see the Supporting Information, S5). Despite a number of attempts
at crystallization, C20 systematically turned out as
a solid not suitable for XRD analysis. In view of that, we decided
to investigate this issue on the basis of theoretical calculations.
cis-Pt Versus trans-Pt Configuration
Tentative Assignment
Density functional theory (DFT) calculations
were performed to evaluate the relative stability of the two isomers.
The relative energies of both isomers were calculated for complex C21 (Figure ) as a model by methods based on quantum mechanics. As a starting
point, the aminothiolated chains were assumed to coordinate by the
sulfur atoms, based on XRD analysis for one of our previous analogs.[11]
Figure 3
Configurations of the metal center in model complex C21.
Configurations of the metal center in model complex C21.Considering K2PtCl4 as the source of Pt(II),
the trans effect seems to point to the trans configuration as the most favorable, as thioether-type ligands are
rated as trans-director when compared to chloride
ligands. With this in mind, the method of choice was the use of Gaussian
program in its 2016 version (abbreviated as Gaussian 16).[18] DFT calculations were performed with the LANL2DZ[19] base and the M06-2X[20] function. Structures were built with the help of graphic program
GaussView,[21] and their energies were optimized
using the values defined by default in the program. Vibration frequencies
were calculated for all compounds, always obtaining zero imaginary
frequencies, which ensures that energy minima are considered.[22]The complexation of sulfur atoms to the
Pt(II) center causes them
to become stereogenic and, therefore, may have the R and/or S configuration. The values of the obtained
energies are shown in Table , together with the energy differences between the cis and trans isomers. The descriptors R and S refer to the absolute configuration
of the sulfur atoms complexed with platinum (see the Supporting Information, Figure S1).
Table 2
Energetic Results
of DFT Calculations
with Gaussian16, M062X/LANL2DZ for cis- and trans-C21 Considering All Possible Absolute
Configurations for Sulfur Atoms[23]
entry
isomera
cis-C21E (hartree)
trans-C21E (hartree)
Δ(cis–trans)b kcal/mol
Δ(trans–trans)b kcal/mol
1
R″/S‴
–1359.7071453
–1359.7283893
13.3
0.0
2
R″/R‴
–1359.7103260
–1359.7218970
11.3
4.1
3
S″/S‴
–1359.7082496
–1359.7247720
12.6
2.3
4
S″/R‴
–1359.7126554
–1359.7253195
9.9
1.9
The configuration descriptors refer
to the sulfur atom of the chain with double prime numbering (′′),
and the chain with triple prime (′′′), as shown
in Figure .
Relative energy values referred
to the most stable trans isomer.
The configuration descriptors refer
to the sulfur atom of the chain with double prime numbering (′′),
and the chain with triple prime (′′′), as shown
in Figure .Relative energy values referred
to the most stable trans isomer.The results in Table indicate that structures with
a trans-configuration
at the platinum center, trans-C21, are
more stable than those for cis-C21 at
about 10–13 kcal/mol. Thus, by extension, it was assumed that
all the complexes here presented might display a trans configuration. Since, for these complexes, it seems that both the
results of the DFT calculations and the prediction based on the trans effect (i.e., thermodynamics and kinetics) point in
the same direction, it would be reasonable to tentatively assign these
complexes a trans configuration.The relative
differences of the corresponding trans-C21 isomers are also given. Among trans-2 isomers, the most stable seems to be R″/S‴ (Table , entry 1), although a more complete conformational
study would be needed to ensure this. The energy differences between
the plausible configurations for trans-C21 are less significant (less than 4.1 kcal/mol), and given the size
of the Pt(II) metallocycle, very likely small conformational changes
would alter the stability order.
Photochemistry
Once the ligands and complexes were
synthesized and characterized, their photochemical behavior was evaluated.
First, the UV–vis spectra of both ligands and complexes were
recorded (see the Supporting Information, Figure S2), whose absorption was found to be clearly bathochromically
shifted relative to our previously reported squaramide-type compounds.[11] Thus, ligand L1 and the corresponding
complex C1 showed spectral maxima at λmaxabs ∼ 380
nm because of the introduction of a pyrrole substituent to the semisquaraine
core. More interestingly, an extension of the conjugation path in
the rest of the compounds by attaching an additional phenylvinyl group
led to a further absorption redshift, which clearly falls in the visible
range (λmaxabs ∼ 450 nm). Therefore, these results validate our strategy
for the design of Pt(II) photocages that are excitable with visible
light.Illumination of the ligands and complexes prepared resulted
in a negligible fluorescence emission regardless of the solvent used
(see the Supporting Information, Table S1). Instead, concomitant irreversible changes were observed in absorption
that suggest that photodegradation of these compounds occurs under
irradiation. This process was investigated in detail upon photoexcitation
at 365 nm for C1 or 450 nm for C2–8 (Figure ). Interestingly,
while for C2–C6 the use of a cosolvent was needed,
complexes C7 and C8 bearing PEG chains could
be solubilized in pure water without the need for any additional cosolvent.
For these and the rest of the compounds, very similar trends in absorption
were registered upon continuous irradiation: the characteristic band
at λmaxabs ∼ 380 nm or λmaxabs ∼ 450 nm rapidly faded after a few
minutes, which in most of the cases resulted in the appearance of
a low-intensity hypsochromically shifted signal (e.g., complexes C2–8 in Figure ). Interestingly, this behavior is reminiscent of that previously
observed by us for squaramide-based Pt(II) complexes,[11] which could be unambiguously ascribed to the photodegradation
of the ligand.
Figure 4
Time evolution of the UV–vis absorption spectra
after irradiation
of complexes (a) C1 in water/DMSO, 98:2, (b) C2 in water/DMF, 98:2, (c) C3 in water/DMSO, 98:2, (d) C4 in water/DMF, 98:2, (e) C5 in water/DMSO,
98:2, (f) C6 in water/DMF, 98:2, (g) C7 in
water, and (h) C8 in water. For C1 λexc = 365 nm (UV lamp) and for C2–8 λexc = 450 nm (LED).
Time evolution of the UV–vis absorption spectra
after irradiation
of complexes (a) C1 in water/DMSO, 98:2, (b) C2 in water/DMF, 98:2, (c) C3 in water/DMSO, 98:2, (d) C4 in water/DMF, 98:2, (e) C5 in water/DMSO,
98:2, (f) C6 in water/DMF, 98:2, (g) C7 in
water, and (h) C8 in water. For C1 λexc = 365 nm (UV lamp) and for C2–8 λexc = 450 nm (LED).To assess the efficiencies of the photodegradation process for
all the ligands and complexes, their photoreaction quantum yields
(Φph) were evaluated (see the Supporting Information, Table S2). In general, the values obtained are
rather low (Φph ∼ 10–4 to
10–5) though they are on the same order of magnitude
as those reported for other visible light-active photolyzable groups.[24] It must be noted that this effect is partially
counterbalanced by the large molar extinction coefficients of our
compounds (εmaxabs > 2.0 × 104 M–1 cm–1, Table S2), as the efficacy
of photoreactions strictly depends on the product of Φph with the absorptivity of the irradiated molecule at the excitation
wavelength. As a result, they present large enough Φph εmaxabs values for photouncaging applications (Table S2). This is especially true for the water-soluble Pt(II) complexes
developed herein, which show Φph and Φph εmaxabs values that are about 10-fold larger than for the corresponding
free ligands and, therefore, make them especially suitable for light-induced
Pt(II) release. Most probably, this is due to the restriction of conformational
mobility upon metal complexation, which must decrease the efficiency
of excited state relaxation through intramolecular vibrational and
rotational motions.At this point, knowing the nature of the
photoproducts and giving
a mechanistic explanation for their formation was considered as required.
Initially, photodegradation of the ligands was explored. First, a
60 mM (DMF/H2O, 9:1) solution of ligand L2 was irradiated (450 nm LED) until its UV–vis spectra revealed
complete degradation, and the resulting mixture was analyzed by MS
and NMR. Three main peaks were detected and correlated with the mass
corresponding to L2 + H2O {[M + H]+ (489.2 Da), [M + Na]+ (511.2 Da) and [M + K]+ (527.2 Da)}. Despite several purification attempts, the NMR analysis
turned out to be too intricate to elucidate its structure. Therefore,
as a structurally simpler model, ligand L22 was prepared
on purpose through a synthetic route similar to that of its analogs
(Scheme ).
Scheme 4
Synthesis
of Simplified Ligand L22
We hypothesized that the photodegradation products would likely
derive from bisketene intermediates, which are known to be formed
upon irradiation of cyclobutenediones.[25] With this idea in mind, the irradiation of L22 was
assayed in DMF/EtOH (6/4) instead of DMF/water, considering that the
addition of the nucleophilic solvent to the ketene functionality would
give us more information (particularly for NMR analysis) if an ethoxy
moiety, instead of a hydroxy, was incorporated to the final degradation
product. The irradiation experiment was monitored by UV spectroscopy
(see the Supporting Information, Figure S3). After irradiation, a major photoproduct was detected by HRMS [443.2
Da (+H+), 465.2 Da (+Na+) and 481.2 Da (+K+)]. These peaks were correlated with the mass corresponding
to L22 + EtOH (see the Supporting Information, Figure S4). Also, a carbonyl group was confirmed
by IR analysis (see the Supporting Information, Figure S5). After careful 1H NMR and 13C NMR analyses of a pure sample of the major degradation product
(see the Supporting Information, Figures S6 and S7 respectively), one of the two regioisomeric butenolides, 24 or 25, was assigned as the most plausible
structure (Scheme ), consisting of a mixture of 3 different conformers. This hypothesis
was also consistent with a similar process previously described for
bisketenes.[25,26] Ultimately, the structure for
the major photoproduct was assigned by additional NOE and HMBC experiments
of the mixture of conformers. NOE Experiments were performed at 250
K (see the Supporting Information, Figure S8), to get a good resolution of the signals corresponding to each
conformer at play. For one of the conformers, the spectrum showed
an NOE interaction between methyl 2‴ and the NHMe group. For the other two conformers, NOE effects between 2‴
and the SMe group were also noticeable. In addition,
an HMBC experiment (see the Supporting Information, Figure S9) revealed no cross signal C2′-H5. With all
those pieces of evidence, the major photoproduct was assigned as butenolide 25. The formation of 25 was envisioned as triggered
by the nucleophilic attack of the nucleophilic solvent to a ketene
functionality as expected (Scheme ). A careful HRMS analysis of an irradiated sample
of complex C2 suggests that the photodegradation pathway
in Scheme is also
effective from this Pt(II) complex. Hence, following the Pt(II) traceability,
a peak [721.1 Da [C2 + H2O–Cl]+] was detected and assigned to the corresponding butenolide
Pt(II) complex (see the Supporting Information, Figure S10).
Scheme 5
Mechanistic Proposal for the Formation of
Butenolide 24 or 25 as the Major Photoproduct
Biological Assessment
Prior to initiating
the evaluation
of the biological activity of the synthesized complexes, a study of
their solubility in an aqueous medium was performed. It was observed
that, among all, complexes wearing PEG motives, C7 and C8, presented excellent water solubility, as expected and,
therefore, they were finally chosen as the candidates to investigate
their biological properties. Accordingly, complexes C7 and C8 were tested against three cancer cell lines
of different origins without illumination and under blue light (light
dose of 32 J/cm2) (Table ). Thus, HeLa, A2780, and cisplatin-resistant A2780cis
cells were incubated for 24 h with stock solutions of C7 and C8 at different concentrations, and cell viability
was evaluated at 72 h. The effect of the compound cytotoxicity was
expressed as a decrease in cell viability (Figure ),[27] and the half
inhibitory compound concentration (IC50) values were also
calculated (Table ).
Table 3
IC50 Determination of C7, C8 Complexes and Cisplatin after 72 h (24
h Internalization) in Light and Dark in HeLa, A2780, and A2780cis
Cells
IC50 (μM)
entry
complex
cell line
lighta
dark
PI
1
C7
HeLa
4 ± 3
>200
>50
2
C7
A2780
1.4 ± 0.8
18 ± 9
13
3
C7
A2780cis
2.7 ± 0.7
19 ± 8
7
4
C8
HeLa
>50
>200
n.d.
5
C8
A2780
5 ± 3
>200
>40
6
C8
A2780cis
48 ± 22
>200
4
7
cisplatin
HeLa
15.5 ± 3.4
14.7 ± 2.6
8
cisplatin
A2780
2.3 ± 0.3
2.8 ± 0.2
9
cisplatin
A2780cis
15.1 ± 1.5
13.8 ± 0.2
λexc = 450 nm and
dose ca. 32 J/cm2. PI = photoactive index (obtained by
dividing the baseline IC50 value by the photoactivated
IC50 value).
Figure 5
Evaluation of cytotoxicity of compounds C7 and C8 in the absence or presence of blue light (dark/light) in
HeLa, A2780, and A2780cis human carcinoma cells after 72 h (24 h internalization)
using PrestoBlue assay. Results are representative of three independent
experiments with a minimum of three replicates per experiment.
Evaluation of cytotoxicity of compounds C7 and C8 in the absence or presence of blue light (dark/light) in
HeLa, A2780, and A2780cis human carcinoma cells after 72 h (24 h internalization)
using PrestoBlue assay. Results are representative of three independent
experiments with a minimum of three replicates per experiment.λexc = 450 nm and
dose ca. 32 J/cm2. PI = photoactive index (obtained by
dividing the baseline IC50 value by the photoactivated
IC50 value).In the absence of light, C7 displayed considerable
cytotoxicity toward A2780 and A2780cis cell lines (IC50 of 18 ± 9 and 19 ± 8 μM, respectively) (entries
2 and 3), while in the same conditions, complex C8 revealed
no toxicity against any of the cancer cell lines. To our delight,
when the complexes were illuminated, the resulting cytotoxicity increased
significantly in all cases, being the most cytotoxic C7 irradiated within A2780 cell line (IC50 = 1.4 ±
0.8 μM) (entry 2). Remarkably, when comparing the light effect,
both complexes showed noticeable PI (photoactive index) values. In
particular, C7 in HeLa (PI > 50) and C8 in
A2780 (PI > 40) displayed remarkable results (entries 1 and 5),
comparable
or even improving examples for Pt(II) complexes in the PACT literature.[5c,28]Using 100 μM concentration as a reference, the photocytotoxicity
of the ligands was also evaluated and compared with their corresponding
Pt(II) complexes. Although for L7, this concentration
was too high to evaluate the light effect, for L8, it
was observed how the cytotoxicity was also enhanced upon illumination.
Nevertheless, the effect is lower compared with the corresponding
Pt(II) complex C8 (Figure ).
Figure 6
Cytotoxicity assays after 72 h incubation (24 h internalization)
in the light and dark in HeLa, A2780, and A2780cis cells with complex C8 and ligand L8, at 100 μM each.
Cytotoxicity assays after 72 h incubation (24 h internalization)
in the light and dark in HeLa, A2780, and A2780cis cells with complex C8 and ligand L8, at 100 μM each.
Cellular Mechanism of Action of Compounds C7 and C8
DNA Interaction
The interaction of these complexes
with DNA was also investigated. To this aim, 50 μM calf thymus
DNA (ct-DNA) was incubated with different molar ratios (ri) of Pt(II)
complexes C7 and C8 and stock solutions
of their corresponding irradiation products C7′ and C8′ (see the Supporting Information, Figure S11). Interestingly, the CD spectra for
both complexes showed an interaction before and after irradiation.
In their nonirradiated forms, C7 and C8 displayed
a similar behavior with a decrease in both positive and negative bands
of ct-DNA (Figure S11a,b, respectively).
Conversely, the irradiation products, C7′ and C8′, showed a differentiated interaction between them.
Whereas for C7′, the positive band remains almost
unaltered with a considerable decrease in the negative band, for C8′, the positive band decreases, with the negative
one being unaffected (Figure S11c,d, respectively).
Hence, although the complexes seem to change the morphology of the
DNA similarly upon incubation in their nonirradiated form, there is
a different type of interaction when the complexes are irradiated: C7′ causing a perturbation in the helicity of the DNA
and C8′ destabilizing the base stacking. Hypothetically,
these different interactions should lead to different in vitro activities
toward tumor cells.Since the target molecule for most Pt drugs
is primarily the nuclear DNA, once we assessed binding by CD, a specific
determination of Pt in DNA was conducted by extracting DNA from the
exposed A2780 cells. Pt concentration was normalized to the DNA concentration,
which was initially measured spectrophotometrically at 260 nm. Figure S11e shows the obtained percentage of
Pt bound to DNA for the A2780 cell line exposed to C7 and C8 complexes at their respective IC50. In the light of these results, we concluded that Pt incorporation
into DNA is much more efficient in the case of compound C7, about 2-fold compared to C8. This result could also
explain why complex C7 is capable of exerting a greater
cytotoxicity than C8.
Apoptosis Detection
To date, different biological action
mechanisms for Pt(II) PACT-based antitumor agents have been proposed.[29] It is well-established that the biological action
of Pt(II) is ultimately related with DNA binding between the water-activated
drug and two adjacent guanine–cytosine base pairs,[30] generating a Pt-DNA cross-link adduct which
impedes cell propagation.[31] Nevertheless,
proteins can also be platinated, inducing direct cell damage and immune
response activation, inducing a mitochondrial-reactive oxygen species
(ROS) response.[32] The damage in the mitochondrial
membrane is enough to hamper apoptosis after cytochrome c release into the cytosol.[33] In addition
to apoptosis, Pt(II) PACT complexes could potentially lead to cell
death via necrosis, with a combination of both apoptosis and necrosis
involved in the final cell cytotoxic effect. Generally, the prevalent
cell death type is dependent not only on the structure, intracellular
localization, and concentration of the complex but also on the light
dose applied and on cell origin. Thus, further biological investigations
were warranted to gain insights into the molecular mechanisms of the
complexes, which will provide helpful insights into the rational design
of photoactivatable platinum-based antitumor complexes. To verify
the mechanism of cell death induced by C7 and C8, A2780 cells were assayed for apoptosis or late apoptosis/necrosis
detection. Cells were treated for 24 h with C7 and C8 at their corresponding IC50 after light activation
(1.5 and 5 μM, respectively) and then photoactivated (light
dose of 32 J/cm2). After a final 72 h of incubation, cells
were stained with an apoptosis/necrosis assay kit which senses the
phosphatidylserine exposure on cells as a hallmark of apoptosis. Cells
were examined under a fluorescent microscope to differentiate between
apoptotic (red), late apoptotic/necrotic (green), and healthy (blue)
cells (Figure ). The
controls (untreated photoactivated and nonphotoactivated cells) displayed
a physiological level of ca. 10% of apoptotic cells but exhibited
no late apoptosis/necrosis. Similar results were detected for complexes C7 and C8 without photoactivation. More interestingly,
after photoactivation, the proportion of apoptotic cells among the
A2780 cells treated with compound C7 rose to 42% at its
IC50, 1.5 μM (Figure ). Under the same conditions, compound C8 induced apoptosis in 36% of cells. The observed results also showed
a significantly increased level of late/apoptotic/necrotic cells after
photoactivation of the complexes, with a special incidence in the C7 compound (56%).
Figure 7
(a) Necrosis and apoptosis assays of A2780 cells
untreated (control
dark), treated with light only (control light), 1.5 μM C7 (C7 dark), light irradiation in the presence
of 1.5 μM C7 (C7 light), 5 μM C8 (C8 dark), and light irradiation in the presence
of 5 μM C8 (C8 light). Healthy viable
cells were stained with CytoCalcein Violet 450 (blue), late apoptotic/necrotic
cells with DNA nuclear green DCS1 (green), and apoptotic cells with
phosphatidylserine (red). (b) Representative histograms of stained
cells. Results are shown as a percentage of total cells.
(a) Necrosis and apoptosis assays of A2780 cells
untreated (control
dark), treated with light only (control light), 1.5 μM C7 (C7 dark), light irradiation in the presence
of 1.5 μM C7 (C7 light), 5 μM C8 (C8 dark), and light irradiation in the presence
of 5 μM C8 (C8 light). Healthy viable
cells were stained with CytoCalcein Violet 450 (blue), late apoptotic/necrotic
cells with DNA nuclear green DCS1 (green), and apoptotic cells with
phosphatidylserine (red). (b) Representative histograms of stained
cells. Results are shown as a percentage of total cells.The obtained data evaluating the cellular death mechanism
induced
by complexes C7 and C8 present promising
cancer-targeting properties by activation of apoptotic pathways. As
shown, the analyzed carcinoma cell line presented apoptosis only after
photoactivation, allowing a better selectivity toward irradiated cancer
cells, with minimal effect over those kept in the dark.
ROS Generation
Study
As previously mentioned, Pt(II)
PACT-based complexes can be related with an oxidative-dependent mechanism
to trigger cell death. In order to detect the formation of intracellular
ROS in A2780 cells in the presence of the complexes while observing
the effect of the photoactivation, the 2′,7′-dichlorofluorescein
diacetate (DCFDA) assay was performed. In brief, DCFDA is cleaved
and oxidized by intracellular esterases and ROS, generating the fluorescent
compound dichlorofluorescein. Treatment with both complexes followed
by light activation resulted in an elevation in cellular ROS release
when compared to control cells (3-fold increase), highlighting the
significant ROS production capabilities of these two photoactivatable
complexes (Figure ). On the contrary, cells treated with complexes but kept in the
dark or the control molecules used (cisplatin and H2O2) in the dark or after light activation did not show a significant
difference in ROS release. These results are in concordance with the
significant cytotoxicity levels previously found for these complexes
(Table ).
Figure 8
ROS formation
measured with the DCFDA assay in A2780 cells for
cisplatin (CisPt) and complexes C7 and C8 at the corresponding IC50 values for each complex (Table ) after treatment
for 4 h. Results are represented as the percentage over untreated
cells. H2O2 (100 μM) was used as the positive
control.
ROS formation
measured with the DCFDA assay in A2780 cells for
cisplatin (CisPt) and complexes C7 and C8 at the corresponding IC50 values for each complex (Table ) after treatment
for 4 h. Results are represented as the percentage over untreated
cells. H2O2 (100 μM) was used as the positive
control.Thus, upon photoactivation, both
complexes C7 and C8 are capable of inducing
ROS production and cell death through
apoptosis. Having low cytotoxicity in the dark, the present complexes
exert a multimechanistic chemotherapeutic effect that may serve for
a targeted cancer chemotherapy.
Summary and Conclusions
Pursuing the improvement of our previous Pt(II) complexes already
published[11] in reference to the solubility
and the photoresponse wavelength, in this study, we have succeeded
in synthesizing, characterizing, and studying the photochemical properties
of eight new semisquaraine-type Pt(II) complexes. The synthesis of
semisquaraine ligands has been carried out by means of a new straightforward
approach. Also, XRD analysis of ligand L2 revealed a
1,2-semisquaraine-type constitution which has been consequently extended
to all the ligands presented. DFT calculations have allowed us to
propose the trans configuration of the platinum center.
Importantly, by structural modification of the ligands, the corresponding
complexes are photoactive at wavelengths such that they allow their
activation in cell cultures. Photodegradation processes have been
studied. Water-soluble complexes C7 and C8 present quantum yields increased with respect to the corresponding
free ligands L7 and L8. By means of NMR
techniques, it has been also possible to determine the structure of
photoproducts of the presented semisquaraines, concluding that what
is generated are butenolide-type derivatives, thus justifying the
changes observed before and after irradiation.Among all the
complexes, two have been selected for their biological
evaluation, mainly because both have an excellent solubility in physiological
media. Thus, complexes C7 and C8 have been
demonstrated to behave as promising photoactivatable compounds in
the presented cancer cell lines, showing a potential amelioration
over cisplatin-resistance in A2780cis cells. Upon photoactivation,
both complexes were capable of inducing a higher cytotoxic effect
on the tested cells compared with nonphotoactivated compounds. Among
the observed results, it is remarkable to note that C7 showed a PI > 50 in HeLa cells and C8 showed a PI
>
40 in A2780 cells, being also effective over cisplatin-resistant A2780cis
cells (PI = 7 and PI = 4, respectively), a finding consistent with
the previously reported Pt(II)-based compounds. Moreover, after photoactivation,
these complexes were also shown to interact with DNA (PACT-likely)
and further cause ROS production (PDT-likely) and ultimately cell
death through apoptosis in the A2780 cancer cell line. We envision
this dual effect[34] as an opportunity to
overcome one of the current PDT drawbacks, as PDT is based on the
conversion of ground-state triplet into excited-state singlet oxygen.
Photo-activated platinum complexes would not require the presence
of oxygen, a potential advantage since tumor cells are often hypoxic.Having a low cytotoxicity in the dark, complexes C7 and C8 may serve as a prodrug that upon photoactivation
in a targeted cancer tissue, exert a potent multimechanistic chemotherapeutic
effect. The controlled photoactivation of these platinum complexes
could allow a targeted cell death in regions of cancer growth and
avoidance of toxic effects on normal cells, and thus may be considered
a potential lead molecule for a targeted cancer chemotherapy.In all, we have been able to design, synthesize, and characterize
new platinum complexes that have been shown to have a powerful cytotoxic
activity, solving problems concerning their solubility and their photoresponse
range. Ultimately, we hope that this study contributes to the development
of new cytotoxic Pt(II) photoactive photocages. More studies in this
line continue to be completed.
Experimental Section
Materials
K2PtCl4 was purchased
from Strem Chemicals. Different organic reagents used for ligands
synthesis were purchased from Sigma-Aldrich or Alfa Aesar. Organic
solvents were dried before use when required. Compounds 9,[14]10,[13]14,[15]15,[16] and 16(17) were synthesized according to previous descriptions.
A solution of 12 (2.13 g, 22.4 mmol)
in anhydrous DMF (10 mL) was added dropwise to a suspension of NaH
(60% in mineral oil, 887 mg, 22.2 mmol) in anhydrous DMF (10 mL) previously
cooled to 0 °C. The resulting suspension was stirred under argon
for 30 min, and (3-bromopropyl)(methyl)sulfane[35] (3.73 g, 22.1 mmol) was added dropwise. The suspension
was stirred at room temperature for 4 h and then poured into water
(40 mL). The mixture was extracted with Et2O (3 ×
20 mL), and each extract was washed with water (3 × 10 mL). The
combined organic layers were dried over anhydrous Na2SO4 and filtered, and the solvent was removed under reduced pressure
to give a yellow oil. The product was purified by column chromatography
(silica gel, hexane/Et2O, 9:1 to hexane/Et2O,
4:1) to give 13 (3.23 g, 17.6 mmol, 80%) as a yellow
oil. IR (ATR): 3105, 2915, 2801, 2721, 1657, 1526, 1480, 1403, 1369,
1321, 1264, 1217, 1078, 1031, 957, 887 cm–1. 1H NMR (400 MHz, CDCl3): δ 7.26 (d, J = 1.1 Hz, 1H), 6.99–6.97 (ddd, J = 2.5 Hz, 1.7 Hz, 1.1 Hz, 1H), 6.94 (dd, J = 4.0
Hz, 1.7 Hz, 1H), 6.22 (dd, J = 4.0 Hz, 2.5 Hz, 1H),
4.42 (t, J = 6.8 Hz, 2H), 2.43 (t, J = 6.8 Hz, 2H), 2.09 (s, 3H), 2.05 (qn, J = 6.8
Hz, 2H). 13C NMR (101 MHz, CDCl3): δ 179.4,
131.8, 131.4, 125.2, 109.7, 47.7, 30.9, 30.1, 15.5. HRMS (ESI+): calcd for [C9H13NOS]: 184.0791 [M
+ H]+, 206.0610 [M + Na]+; found, 184.0795 [M
+ H]+, 206.0616 [M + Na]+.
A suspension of NaH (60% dispersion in mineral
oil, 51.7 mg, 1.29 mmol) in anhydrous DMF (0.5 mL) was cooled to 0
°C under an argon atmosphere. Then, a solution of diethyl (3,4,5-tris{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}benzyl)phosphonate[36] (431.6 mg, 0.60 mmol) in anhydrous DMF (0.7
mL) was added dropwise. The reaction mixture was stirred at 0 °C
for 30 min, then a solution of 13 (112.8 mg, 0.62 mmol) in anhydrous
DMF (0.7 mL) was added, and the mixture was heated at 100 °C
for 16 h. After this time, the mixture was cooled to 0 °C, and
H2O (5 mL) was added dropwise. The aqueous phase was extracted
with EtOAc (3 × 5 mL). Each organic phase was washed with brine
(3 × 5 mL). The combined organic extracts were dried over anhydrous
Na2SO4 and filtered. The solvent was removed
under reduced pressure, and the residue was purified by column chromatography
(silica gel, EtOAc to EtOAc/MeOH, 95:5) to give crude 19 (341.2 mg) as a dark brown oil, which was used in the next step
without further purification.A solution of crude 19 (238.8 mg, 0.32 mmol) in CH2Cl2 (3 mL) was
added dropwise to a mixture of a solution of dichlorocyclobutenedione 9 (49.9 mg, 0.33 mmol) in CH2Cl2 (7
mL) and H2O (2 mL). The organic layer turned dark red,
and the mixture was stirred at room temperature for 1 h. After this
time, a solution of 11 (36 μL, 0.32 mmol) and DIPEA
(113 μL, 0.64 mmol) in CH2Cl2 (1 mL) was
added. The resulting mixture was stirred at rt for 90 min. Then, the
organic layer was dried over anhydrous Na2SO4 and filtered. The solvent was removed under reduced pressure, and
the residue was purified by column chromatography (silica gel, EtOAc
to EtOAc/MeOH, 95:5) to give pure L7 (245.6 mg, 0.26
mmol, 62% over 2 steps) as a red-orange oil. The oil was kept in the
freezer turning into a brown sticky solid. mp 46–49 °C
(from CH2Cl2). IR (ATR): 2919, 2876, 1766, 1714,
1596, 1533, 1504, 1466, 1431, 1341, 1295, 1270, 1109, 952, 854 cm–1. 1H NMR (400 MHz, CDCl3): δ
7.00 (d, Jtrans = 16.0 Hz, 1H), 6.92 (d, Jtrans = 16.0 Hz, 1H), 6.75 (s, 2H), 6.66 (d, J = 4.3 Hz, 1H), 6.53 (t, J = 6.5 Hz, 1H),
6.49 (d, J = 4.3 Hz, 1H), 4.75 (t, J = 6.9 Hz, 2H), 4.22–4.13 (m, 6H), 3.99 (q, J = 6.5 Hz, 2H), 3.89–3.49 (m, 30H), 3.36 (s, 3H), 3.36 (s,
6H), 2.66 (t, J = 6.5 Hz, 2H), 2.51 (t, J = 6.9 Hz, 2H), 2.13 (s, 3H), 2.06 (s, 3H), 2.05–1.95 (m,
4H). 13C NMR (100 MHz, CDCl3): δ 188.7,
185.4, 176.8, 156.5, 153.0, 139.1, 138.6, 132.5, 130.8, 125.2, 115.3,
112.6, 109.4, 106.5, 72.6, 72.1, 70.9, 70.8, 70.7, 70.6, 69.9, 69.1,
59.1, 45.1, 44.9, 31.9, 31.4, 31.3, 29.5, 15.8, 15.6. HRMS (ESI+): calcd for [C45H70N2O14S2]: 927.4341 [M + H]+, 949.4161 [M
+ Na]+; found, 927.4326 [M + H]+, 949.4164 [M
+ Na]+. UV (H2O) λmax, nm (ε,
M–1 cm–1): 212 (2.15 × 104), 235 (1.70 × 104), 308 (1.03 × 104), 432 (4.06 × 104).
A solution of 17 (88.1 mg, 0.31
mmol) in CH2Cl2 (3 mL) was added dropwise to
a mixture of a solution of dichlorocyclobutenedione 9 (46.5 mg, 0.31 mmol) in CH2Cl2 (6 mL) and
H2O (2 mL). The organic layer turned dark red, and the
resulting mixture was stirred at room temperature under argon for
1 h. After this time, a solution of methylamine (33% wt in MeOH, 38
μL, 0.31 mmol) and DIPEA (105 μL, 0.60 mmol) in CH2Cl2 (2 mL) was added, and the mixture was stirred
for 30 min. Then, the organic layer was separated, dried over anhydrous
Na2SO4, and filtered. The resulting filtrate
was cooled to 0 °C, and Et2O was added. An orange
solid precipitated, which was filtered and washed with Et2O to give pure L22 (45 mg, 0.11 mmol, 37%) as an orange
powder. mp >220 °C (from CH2Cl2/Et2O). IR (ATR): 3293, 2910, 1766, 1699, 1569, 1507, 1450, 1426,
1386, 1294, 1251, 1172, 1130, 1110, 1026, 956, 816 cm–1. 1H NMR (400 MHz, DMSO-d6): δ 8.43 (q, J = 4.5 Hz, 1H), 7.61–7.53
(m, 2H), 7.17 (d, J = 16.2 Hz, 1H), 7.12 (d, J = 16.2 Hz, 1H), 7.00–6.92 (m, 2H), 6.89 (d, J = 4.3 Hz, 1H), 6.83 (d, J = 4.3 Hz, 1H),
4.75 (t, J = 7.4 Hz, 2H), 3.78 (s, 3H), 3.31 (d, J = 4.5 Hz, 3H), 2.38 (t, J = 7.4 Hz, 2H),
1.99 (s, 3H), 1.82 (qn, J = 7.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 189.4,
184.3, 175.9, 159.1, 155.1, 138.3, 129.6, 127.9, 124.7, 114.2, 114.0,
113.9, 108.8, 55.2, 44.2, 31.3, 29.9, 14.6. HRMS (ESI+):
calcd for [C22H24N2O3S]:
397.1580 [M + H]+, 419.1400 [M + Na]+; found,
397.1567 [M + H]+, 419.1385 [M + Na]+. UV (DMF)
λmax, nm (ε, M–1 cm–1): 302 (1.48 × 106), 429 (6.08 × 106), 446 (6.44 × 106).
Synthesis of Complexes
Complex C1
Ligand L1 (86.7
mg, 0.26 mmol) and K2PtCl4 (105.6 mg, 0.25 mmol)
were suspended in a mixture of MeOH and water (1:1, 4 mL). The mixture
was stirred at room temperature under an argon atmosphere for 17 h.
After this time, a brown solid precipitated. After filtration, the
solid was washed with hot MeOH and water and then dried to afford C1 as a brown powder (82.0 mg, 0.14 mmol, 56%). mp 173–178
°C (from MeOH/H2O, 1:1). IR (ATR): 3283, 2920, 1772,
1706, 1589, 1537, 1478, 1421, 1355, 1264, 1132, 1092, 970 cm–1. 1H NMR (400 MHz, DMSO-d6) as a mixture of products due to one Cl exchange: 8.67–8.51
(m), 7.31–7.16 (m), 6.92–6.81 (m), 6.40–6.26
(m), 4.61 (t, J = 6.9 Hz), 4.54 (t, J = 7.0 Hz), 3.86–3.71 (m), 2.59–2.50 (bs), 2.36–2.28
(bs), 1.93–1.81 (bs), 2.21–1.96 (bs). 13C
NMR (101 MHz, DMSO-d6) as a mixture of
products due to one Cl exchange: δ 190.0 + 189.8, 184.8 + 184.7,
176.0, 156.4, 156.2, 128.9, 123.3, 114.0–113.7, 110.6–110.4,
48.0, 47.6, 43.5, 43.1, 35.4, 35.3, 31.4, 30.2, 30.0, 29.9, 29.7,
28.4, 21.0, 20.6. HRMS (ESI+): calcd for [C16H22N2O2S2PtCl2]: 532.0688 [M–2Cl–H]+, 569.0444 [M–Cl]+, 605.0203 [M + H]+, 610.0827 [M–2Cl–H
+ DMSO]+, 647.0582 [M–Cl + DMSO]+; found,
532.0678 [M–2Cl–H]+, 569.0443 [M–Cl]+, 605.0206 [M + H]+, 610.0817 [M–2Cl–H
+ DMSO]+, 647.0583 [M–Cl + DMSO]+. EA
calcd for C16H22N2O2S2PtCl2 (%): C, 30.87; H, 3.89; N, 4.50; S, 10.30
[M + H2O]. Found: C, 30.76; H, 3.49; N, 4.22; S, 10.12
[M + H2O]. UV (DMSO) λmax, nm (ε,
M–1 cm–1): 276 (1.40 × 104), 366 (2.25 × 104), 445 (9.32 × 102), 516 (2.30 × 102).
Complex C2
A solution of K2PtCl4 (153.9 mg,
0.37 mmol) in H2O (5 mL) was
added to a solution of L2 (172.0 mg, 0.37 mmol) in THF
(5 mL). The solution was stirred at room temperature under argon for
22 h. After this time, an orange solid precipitated. After filtration,
the solid was washed with THF and water and dried to afford C2 as an orange powder (262.2 mg, 0.36 mmol, 97%). mp 184–187
°C (from THF/H2O). IR (ATR): 3302, 2916, 1764, 1701,
1584, 1533, 1509, 1461, 1425, 1299, 1247, 1174, 1141, 1029, 957, 850,
816 cm–1. 1H NMR (400 MHz, DMF-d7): δ 8.75–8.51 (m), 7.75–755
(m), 7.40–7.65 (m), 5.06–4.63 (m), 4.23–3.76
(m), 3.41–2.83 (m), 2.69–2.02 (m). HRMS (ESI+): calcd for [C25H30N2O3S2PtCl2]: 737.0781 [M + H]+, 759.0601
[M + Na]+, 775.0339 [M + K]+; found, 737.0791
[M + H]+, 759.0607 [M + Na]+, 775.0353 [M +
K]+. EA calcd for C25H30N2O3S2PtCl2 (%): C, 40.76; H, 4.11;
N, 3.80; S, 8.70. Found: C, 40.96; H, 4.33; N, 3.51; S, 8.55. UV (DMF)
λmax, nm (ε, M–1 cm–1): 305 (1.61 × 104), 430 (6.18 × 104), 446 (5.95 × 104).
Complex C3
A solution of K2PtCl4 (89.2 mg, 0.22
mmol) in H2O (3 mL) was
added to a solution of L3 (95.1 mg, 0.21 mmol) in THF
(3 mL). The solution was stirred at room temperature under argon for
15 h. After this time, a brown-red solid precipitated. After filtration,
the solid was washed with H2O, MeOH, and THF and dried
to afford C3 as a brown-red powder (130.8 mg, 0.18 mmol,
86%). mp >200 °C (from THF/H2O). IR (ATR): 1736,
1691,
1579, 1532, 1510, 1461, 1421, 1267, 1168, 1138, 1044, 955, 850, 810
cm–1. 1H NMR (400 MHz, DMSO-d6): δ 9.65 (s), 8.70–8.51 (m), 7.59–7.41
(m), 7.22–6.62 (m), 4.92–4.63 (m), 3.91–3.68
(m), 3.27–3.03 (m), 2.70–2.24 (m), 2.15–1.70
(m). MS (ESI+): calcd for [C24H28N2O3S2PtCl2]: 687.1 [M–Cl]+, 745.0 [M + Na]+, 765.1 [M–Cl + DMSO]+; found, 687.1 [M–Cl]+, 745.0 [M + Na]+, 765.1 [M–Cl + DMSO]+. EA calcd for C24H28N2O3S2PtCl2 (%): C, 38.45; H, 4.17; N, 3.74; S, 8.55 [M + 3/2H2O]. Found: C, 38.39; H, 3.82; N, 3.50; S, 8.27 [M + 3/2H2O]. UV (DMSO) λmax, nm (ε, M–1 cm–1): 303 (1.58 × 104), 436 (5.22
× 104), 450 (5.29 × 104).
Complex C4
A solution of K2PtCl4 (57.2
mg, 0.14 mmol) in water (1.5 mL) was added
to a solution of L4 (69.6 mg, 0.13 mmol) in THF (1.5
mL). The solution was stirred at room temperature under argon for
20 h. After this time, an orange solid precipitated. After filtration,
the solid was washed with THF and water and dried to afford C4 as an orange powder (102.9 mg, 99%). mp >200 °C
(from
THF/H2O). IR (ATR): 3299, 2930, 1762, 1701, 1578, 1529,
1503, 1459, 1416, 1336, 1266, 1241, 1184, 1120, 1041, 997, 951, 808,
760 cm–1. 1H NMR (400 MHz, DMF-d7): δ 8.74–8.57 (m), 7.52–7.14
(m), 7.13–6.96 (m), 6.95–6.79 (m), 5.07–4.80
(m), 4.07–3.85 (m), 3.81–3.72 (m), 3.69–3.60
(m), 3.37–2.83 (m), 2.70–2.05 (m), 1.88–1.74
(m). HRMS (ESI+): calcd for [C27H34N2O5S2PtCl2]: 797.0994
[M + H]+, 819.0813 [M + Na]+, 835.0551 [M +
K]+; found, 797.0978 [M + H]+, 819.0798 [M +
Na]+, 835.0551 [M + K]+. EA calcd for C27H34N2O5S2PtCl2 (%): C, 39.81; H, 4.45; N, 3.44; S, 7.87 [M + H2O]. Found: C, 39.42, H, 4.15, N, 3.15, S, 7.49 [M + H2O]. UV (DMF) λmax, nm (ε, M–1 cm–1): 307 (1.31 × 104), 429 (6.40
× 104), 448 (6.18 × 104).
Complex C5
A solution of K2PtCl4 (54.8
mg, 0.13 mmol) in H2O (2 mL) was
added to a brown suspension of L5 (63.8 mg, 0.13 mmol)
in THF (2 mL). The solution was stirred at room temperature under
argon for 16 h. After this time, a brown solid precipitated. After
filtration, the solid was washed with H2O, MeOH, and THF
and dried to afford C5 (80.5 mg, 0.11, 85%) as a brown
solid. mp >200 °C (from THF/H2O). IR (ATR): 3317,
1767, 1699, 1585, 1526, 1462, 1426, 1311, 1187, 1139, 1032, 998, 958,
819 cm–1. 1H NMR (400 MHz, DMSO-d6): δ 8.98–8.81 (m), 8.69–8.35
(m), 7.11–6.75 (m), 6.65–6.51 (m), 4.89–4.64
(m), 3.93–3.57 (m), 3.44–3.07 (m), 2.65–1.62
(m). MS (ESI+): calcd for [C24H28N2O5S2PtCl2]: 719.1 [M–Cl]+, 755.1 [M + H]+, 777.0 [M + Na]+, 797.1
[M–Cl + DMSO]+; found, 719.1 [M–Cl]+, 755.0 [M + H]+, 777.0 [M + Na]+, 797.1 [M–Cl
+ DMSO]+. EA calcd for C24H28N2O5S2PtCl2 (%): C, 37.31;
H, 3.91; N, 3.63; S, 8.30 [M + H2O]. Found: C, 37.14, H,
3.88, N, 3.31, S, 7.67 [M + H2O]. UV (DMSO) λmax, nm (ε, M–1 cm–1): 306 (1.28 × 104), 439 (5.62 × 104), 455 (5.78 × 104).
Complex C6
A solution of K2PtCl4 (67.0 mg, 0.16
mmol) in H2O (2 mL) was
added to a solution of L6 (96.3 mg, 0.16 mmol) in THF
(2 mL). The resulting mixture was stirred at room temperature under
argon for 16 h. After this time, a sticky orange solid precipitated,
which was filtrated and washed with H2O, MeOH, and THF
and dried. The sticky solid was digested with Et2O to give
pure C6 (111.3 mg, 0.13 mmol, 81%) as an orange powder.
mp 135–139 °C (from THF/H2O). IR (ATR): 2869,
1762, 1704, 1583, 1533, 1509, 1460, 1424, 1371, 1298, 1236, 1176,
1138, 1093, 1057, 954, 849, 817 cm–1. 1H NMR (400 MHz, DMSO-d6): δ 8.65–8.51
(m), 7.66–7.51 (m), 7.25–7.08 (m), 7.02–6.80
(m), 4.93–4.66 (m), 4.23–4.04 (m), 3.88–3.68
(m), 3.65–3.48 (m), 3.47–3.40 (m), 3.26–3.20
(m), 2.92–2.75 (m), 2.61–2.34 (m), 2.18–2.03
(m), 2.01–1.96 (m), 1.95–1.78 (m). MS (ESI+): calcd for [C31H42N2O6S2PtCl2]: 869.2 [M + H]+, 891.1
[M + Na]+; found, 869.2 [M + H]+, 891.1 [M +
Na]+. EA calcd for C31H42N2O6S2PtCl2 (%): C, 41.99, H, 5.00,
N, 3.16, S, 7.23 [M + H2O]. Found: C, 42.07, H, 4.79, N,
3.14, S, 7.15 [M + H2O]. UV (DMF) λmax, nm (ε, M–1 cm–1): 304
(1.90 × 104), 429 (7.19 × 104), 446
(6.93 × 104).
Complex C7
A solution of K2PtCl4 (109.6 mg,
0.26 mmol) in H2O (4 mL) was
added to a solution of L7 (250.4 mg, 0.26 mmol) in THF
(4 mL). The solution was stirred at room temperature under argon for
17 h. After this time, 20 mL of brine was added, and the product was
extracted with CH2Cl2 (3 × 20 mL). The
combined organic layers were dried over anhydrous Na2SO4 and filtrated. The product was recrystallized in CH2Cl2/Et2O to give C7 (207.2 mg,
0.17 mmol, 65%) as a brown solid. mp 92–95 °C (from CH2Cl2/Et2O). IR (ATR): 2874, 1764, 1707,
1590, 1530, 1503, 1459, 1429, 1338, 1248, 1096, 951.92, 851 cm–1. 1H NMR (400 MHz, CDCl3): δ
7.12–6.50 (m), 4.44–4.04 (m), 3.97–3.44 (m),
3.39–3.30 (m), 2.62–2.01 (m), 1.82 (s). MS (ESI+): calcd for [C45H70N2O14S2PtCl2]: 1215.3 [M + Na]+; found, 1215.3 [M + Na]+. EA calcd for C45H70N2O14S2PtCl2 (%): C, 42.64; H, 5.57; N, 2.21; S, 5.06 [M + KCl]. Found: C, 42.73,
H, 5.58, N, 2.11, S, 4.82 [M + KCl]. UV (H2O) λmax, nm (ε, M–1 cm–1): 221 (3.19 × 104), 237 (2.83 × 104), 312 (1.11 × 104), 432 (3.66 × 104), 462 (2.71 × 104).
Complex C8
A solution of K2PtCl4 (235.5 mg,
0.57 mmol) in H2O (6 mL) was
added to a solution of L8 (Mw = 2439, 1.06 g, 0.43 mmol) in H2O (7 mL). The solution
was stirred at room temperature under argon for 16 h. After this time,
brine (20 mL) was added, and the mixture was extracted with CH2Cl2 (3 × 20 mL). The combined organic extracts
were dried over anhydrous Na2SO4 and filtered,
the solvent was removed under reduced pressure, and the residue was
recrystallized from CH2Cl2/Et2O to
give C8 (1.10 g, 0.41 mmol, 95%) as a brown solid. mp
50–53 °C (from CH2Cl2/Et2O). IR (ATR): 2878, 1765, 1706, 1596, 1510, 1466, 1342, 1279, 1241,
1145, 1100, 1060, 961, 842 cm–1. 1H NMR
(400 MHz, CDCl3): δ 7.76–6.45 (m), 4.99–3.48
(m), 3.46–3.40 (m), 3.36 (s), 2.79–2.00 (m). ICP-OES:
6.7% Pt. UV (H2O) λmax, nm (ε, M–1 cm–1): 235 (2.86 × 104), 308 (1.36 × 104), 434 (2.75 × 104), 462 (2.18 × 104).
Complex C20
A solution of K2PtCl4 (60.9 mg, 0.15
mmol) in H2O (2 mL) was
added to a solution of L20 (98.1 mg, 0.14 mmol) in THF
(2 mL). The solution was stirred at room temperature under argon for
17 h. After this time, 20 mL of brine was added, and the product was
extracted with CH2Cl2 (3 × 20 mL). The
combined organic layers were dried over anhydrous Na2SO4 and filtrated. The product was recrystallized from CH2Cl2/Et2O to give pure C20 (114.0 mg, 0.12 mmol, 86%) as a dark orange solid. mp 190–195
°C (from CH2Cl2/Et2O). IR (ATR):
2929, 2856, 1764, 1702, 1584, 1533, 1506, 1461, 1425, 1253, 1169,
1141, 1104, 955, 910, 851, 819 cm–1. 1H NMR (360 MHz, CDCl3): δ 7.76–7.62 (m),
7.49–7.13 (m), 7.01–6.39 (m), 4.82–4.33 (m),
4.24–3.86 (m), 3.33–1.81 (m), 1.29–0.79 (m).
MS (ESI+): calcd for [C40H46N2O3S2SiPtCl2]: 925.2 [M–Cl]+, 961.2 [M + H]+, 983.2 [M + Na]+; found,
925.2 [M–Cl]+, 961.2 [M + H]+, 983.2
[M + Na]+. EA calcd for C40H46N2O3S2SiPtCl2 (%): C, 49.99;
H, 4.82; N, 2.92; S, 6.67. Found: C, 49.72, H, 4.85, N, 2.72, S, 6.25.
UV (CHCl3) λmax, nm (ε, M–1 cm–1): 310 (9.15 × 103), 429 (3.83
× 104), 455 (3.02 × 104).
X-ray Structure Determination
Crystals of L2 were mounted on a glass fiber and used for the data collection on
a Bruker D8 Venture with a photon detector equipped with graphite-monochromated
Cu Kα radiation (λ = 1.54178 Å). The data reduction
was performed with APEX2[39] software and
corrected for absorption using SADABS.[40] Crystal structures were solved by direct methods using SIR97 program[41] and refined by full-matrix least-squares on F2, including all reflections using anisotropic
displacement parameters by means of WINGX crystallographic package.[42] Generally, anisotropic temperature factors were
assigned to all atoms except for hydrogen atoms, which are riding
their parent atoms with an isotropic temperature factor arbitrarily
chosen as 1.2 times that of the respective parent. Final R(F), wR(F2), the goodness of fit agreement factors, and details on the
data collection and analysis can be found in Table S3. Crystallographic data (excluding structure factors) for
the structure reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publication
Nr. CCDC 2100958 for compound L2. Copies of the data
can be obtained free of charge on application to the Director, CCDC,
12 Union Road, Cambridge, CB2 1EZ, U.K. (Fax: +44-1223-335033; e-mail: deposit@ccdc.cam.ac.uk).
Photochemical Characterization
UV–vis absorption
measurements were recorded on a HP 8453 spectrophotometer.Fluorescence
emission spectra were recorded using a custom-made spectrofluorometer,
where the sample was excited with a cw diode laser (λexc = 405 nm), and emitted photons were detected using an Andor ICCD
camera coupled to a spectrograph. All the emission spectra registered
were corrected by the wavelength dependence of the spectra response
of the detection system. Samples were prepared in spectroscopic grade
solvents and adjusted to a response within the linear range.Fluorescence quantum yields were determined using the standard
method for highly diluted solutions to prevent selfabsorption processes
(absorption <0.1 at λexc) and relative to 9,10-bis(phenylethynyl)anthracene
in acetonitrile (Φfl = 0.985).[43]Continuous irradiation of the solutions of photoactive
compounds
was performed using a UV lamp (Vilber-Lormat, λ = 365 nm, 4
W) or a blue LED (λexc ∼ 450 nm,[26] 3 W).Photodegradation quantum yields
were determined using a reported
methodology[44] and relative to trans-azobenzene in acetonitrile (Φtrans→cis =
0.14)[45] for L1 and C1 [excited with a Nd/YAG (Brilliant, Quantel) pulsed laser emitting
at 355 nm] and to 1,2-bis(5-chloro-2-methyl-3-thienyl-perfluorocyclopentene)
in hexane (Φclosed→open = 0.13)[46] for L2–8 and C2–8 (excited with a diode cw laser with λexc = 445
nm).To characterize the photoproducts, 10–2 M solutions
of L2 (DMF/H2O, 9:1) and L22 (DMF/EtOH,
6:4) and C2 (DMF-d7/D2O, 9:1) were irradiated with a blue LED (λexc ∼ 450 nm,[26] 10 W) until the total
disappearance of the absorption maxima band of the initial species
(monitored by UV–vis measurements of aliquots). For L2 and L22, after total photodegradation, H2O was added, and the crude products were extracted with CH2Cl2. The combined extracts were dried over anhydrous Na2SO4, filtered, and the solvent was removed under
a reduced pressure. Products were purified by column chromatography
(silica gel, hexane/EtOAc 6:4 to hexane/EtOAc, 3:7). In the case of C2, after total photodegradation, the sample was freeze-dried
to remove the solvent.
CD Spectroscopy
CD spectroscopy
was performed using
a model J-715 spectropolarimeter (JASCO, Gross-Umstadt, Germany) equipped
with a computer (J-700 software, JASCO) with 1 cm thick quartz cuvettes.
Measurements were carried out at a constant temperature of 25 °C.
CD spectra were measured in 10 mM Tris-HCl buffer (pH 7.24). The calf
thymus DNA concentration was 50 μM. Different samples with an
increasing amount of the Pt complex to study (0, 25, and 50 μM)
were incubated at 37 °C for 24 h before spectra were recorded
in the range of 200–350 nm. Complexes were added from 10–3 M stock solutions in water/DMSO, 98:2, solvent mixtures.
Irradiated stocks were prepared by irradiation with a blue LED (λexc ∼ 450 nm, 3 W) until the total disappearance of
the absorption maxima band of the initial species (monitored by UV–vis
measurements of aliquots).
Cell Culture
Human ovarian cancer
cells A2780 and human
ovarian cancer cells cisplatin-resistant A2780cis were obtained from
the European Collection of Authenticated Cell Cultures (ECACC, UK)
and were routinely cultured in Dulbecco’s modified Eagle’s
medium (Invitrogen) containing 10% heat-inactivated fetal bovine serum
(FBS) at 37 °C in a humidified CO2 atmosphere. Human
adenocarcinoma cells (HeLa) were obtained from American Type Culture
Collection (ATCC, Manassas, VA, USA) and were routinely cultured in
modified Eagle’s medium alpha (MEM-α, Invitrogen) containing
10% heat-inactivated fetal bovine serum at 37 °C in a humidified
CO2 atmosphere.
DNA Isolation
To investigate the
Pt content associated
with the DNA of A2780 cells, DNA was extracted and purified by using
the phenol/chloroform methodology. In brief, 5 × 106 cells were seeded in 150 mm dishes and allowed to attach overnight.
Cells were then incubated with the different compounds for 24 h in
a 10% serum-containing medium. Following incubation, the supernatant
was discarded, and the cells were washed thoroughly 3 times with 5
mL ice-cold PBS. Cells were trypsinized and transferred into a 15
mL tube for centrifugation at 500g for 5 min. The
obtained pellet was resuspended in lysis buffer (100 mM NaCl, 10 mM
Tris-Cl pH 8, 25 mM EDTA pH 8, 0.5% SDS, 0.1 mg/mL proteinase K),
and genomic DNA was purified by the phenol/chloroform extraction.
The extracted DNA was eluted using 1 mL of the TE buffer (10 mM Tris-Cl
pH 9, 0.1 mM EDTA). The DNA concentration and purity of the sample
were determined by spectral photometry (NanoDrop 2000c, Thermo Scientific)
in triplicate.
ICP–MS
For quantification
of the metal content,
1 mL of the DNA solution was wet-digested by adding a freshly prepared
mixture of concentrated HNO3 at 105 °C for 1 h. Samples
were subsequently dissolved in dilute HNO3 (1%, v/v) before
being analyzed by ICP–MS. The platinum content was determined
by ICP–MS with an Agilent 7900 inductively coupled plasma mass
spectrometer.
Cell Viability Assays
Cell proliferation
was evaluated
using the PrestoBlue assay. Cells were plated in 96-well sterile plates
at a density of 5 × 103 cells per well (100 μL)
and allowed to grow for 24 h. Cells were then incubated with various
concentrations of the studied compounds and cisplatin (0–200
μM for dose–response curves) for 24 h at 37 °C.
The next day, cells were washed three times with a fresh culture medium
to remove noninternalized complex excess, either untreated or light-activated,
for 15 min and left to grow for an additional 48 h at 37 °C.
Following the treatment, 10 μL of the PrestoBlue solution was
added to each well, and the plates were incubated for an additional
2 h at 37 °C. Afterward, fluorescence was measured using a Victor3
(PerkinElmer) fluorescence multiwell plate reader with the excitation/emission
wavelengths set at 531/572 nm. The cell viability was expressed as
percentage values with respect to control cells, and the data are
shown as the mean value ± standard error of the mean (SEM) of
three independent experiments. Dose–response curves and the
corresponding IC50 values were obtained by means of nonlinear
regression (curve fit), calculated with GraphPad Prism 6.0 software.
Apoptosis Detection Assay
Assessment of apoptotic,
late apoptotic/necrotic, and healthy cells with a fluorescence microscope
was performed using the Apoptosis/Necrosis Detection Kit (Abcam 176750).
A2780 cells (104 cells/well) were plated on 96-well plates
and allowed to adhere overnight. Cells were either untreated or treated
with 1.5 μM of complex C7 and 5 μM of complex C8 for 24 h. Cells were washed with a fresh medium and either
untreated or light-activated for 15 min and incubated for a final
time of 72 h. Cells were washed twice with the assay buffer, and then
the staining buffer (containing 5 μL of apopxin deep red indicator,
5 μL of nuclear green, and 5 μL of CytoCalcein 450 to
each 1 mL of the assay buffer) was added and, finally, incubated for
60 min at room temperature. Afterward, the cells were washed three
times with the assay buffer, and a total of 10 random images were
taken using the Cy5 (Ex/Em = 630/660 nm), FITC (Ex/Em = 490/520 nm),
and violet (Ex/Em = 405/450 nm) channels of a fluorescent microscope
at a 20× objective. Cells from images were counted, and the numbers
of each cellular state were recorded.
ROS Production Assay
Measurement of ROS was achieved
using the DCFDA reagent ROS detection assay. A2780 cells were plated
in a 96-well plate at 2 × 104 cells/well in a medium
supplemented with 10% FBS and allowed to adhere overnight. The next
day cells were incubated with DCFDA (100 μL/well of a 25 μM
solution) for 30 min in the dark. Cells were then washed, either untreated
or treated, with cisplatin or complexes C7 and C8 at their IC50 and incubated for an additional
4 h. After this time, cells were washed, fresh medium was added, and
light-activated for 15 min or maintained in the dark to finally be
incubated for a 48 h extra time. The experiments were run in triplicate.
H2O2 was used as a positive control at 100 μM.
The fluorescence of each well was measured in a Microplate Reader
Victor3 (PerkinElmer) at 535 nm after an excitation at 485 nm.
Authors: David C Harrowven; Mubina Mohamed; Théo P Gonçalves; Richard J Whitby; David Bolien; Helen F Sneddon Journal: Angew Chem Int Ed Engl Date: 2012-03-22 Impact factor: 15.336
Authors: Victoria Cepeda; Miguel A Fuertes; Josefina Castilla; Carlos Alonso; Celia Quevedo; Jose M Pérez Journal: Anticancer Agents Med Chem Date: 2007-01 Impact factor: 2.505
Authors: Elisa Bassotti; Paola Carbone; Alberto Credi; Marco Di Stefano; Stefano Masiero; Fabrizia Negri; Giorgio Orlandi; Gian Piero Spada Journal: J Phys Chem A Date: 2006-11-16 Impact factor: 2.781
Authors: Michael A Jakupec; Mathea S Galanski; Vladimir B Arion; Christian G Hartinger; Bernhard K Keppler Journal: Dalton Trans Date: 2007-11-07 Impact factor: 4.569
Figure 1
Scheme 1
Scheme 2
Scheme 3
Figure 2
Figure 3
Figure 4
Scheme 4
Scheme 5
Figure 5
Figure 6
Figure 7
Figure 8